In the field of organ transplantation, it has been known since the 1930s that compatibility between the donor's tissue type, as defined by the HLA (Human Leucocyte Antigen) antigens, and the recipient's immune system, especially the antibodies, is essential to the success of the organ transplant.
The HLA antigens are carried by two types of membrane proteins which are highly immunogenic: HLA class I molecules and HLA class II molecules. Accordingly, the exposure of an individual to HLA alloantigens, that is to say antigens that are foreign and different from his own, can lead to the development of an immune response to those antigens. This immune response can be cell-mediated (alloreactive T lymphocytes) or humoral (synthesis of anti-HLA antibodies).
HLA class I antigens are coded for by three genes HLA-A, HLA-B and HLA-C, the polymorphism of which is responsible for the three series of alleles HLA-A, HLA-B and HLA-C, respectively. HLA class II antigens are coded for by the genes HLA-DP, HLA-DQ and HLA-DR.
In organ transplantation, it is crucial to minimise—or even eliminate—the risks of proposing to a patient awaiting a transplant an organ for transplant that expresses HLA antigens against which the patient is already immunised. In this situation, the risk of the occurrence of hyperacute humoral rejection—that is to say humoral rejection within a period of less than 24 hours following the transplant—is considerable. In addition, within the context of transplant monitoring, the early screening of the appearance in the transplant recipient patient of antibodies directed against the antigens of the transplanted organ allows said transplant recipient patient to be treated as early as possible in an attempt to control the development of the humoral response, which may result in the destruction of the transplant.
Monitoring of the alloimmunisation of both transplant patients and patients awaiting a transplant is therefore essential in order to ensure the survival of the transplants and of the transplant patients.
Within this context, it is necessary to be able to detect, identify and quantify anti-HLA antibodies in patients awaiting a transplant and in transplant patients. Numerous techniques for detecting anti-HLA antibodies have already been developed.
There is known in particular the technique called “complement-dependent microlymphocytotoxicity”. This technique consists in presenting the serum of a patient, especially of a transplant recipient, to a series of cells of known HLA typing in the presence of rabbit complement. If antibodies (Ab) specific to the HLA antigens carried by the cells are present in the tested serum, and if those antibodies are capable of activating the complement (antibodies of class IgM and of subclass IgG-1 and IgG-3), complement-dependent cell lysis (CDC, Complement Dependent Cytotoxicity) reveals the presence of the antibodies. By virtue of a panel of cells expressing different HLA antigens, it is thus possible to screen the antibodies and then identify their specificity/specificities. This reference technique permits the detection of cytolytic anti-HLA antibodies, which are the most dangerous for the transplanted organ. However, this technique has low sensitivity in comparison with more recent techniques. This technique, which requires either the availability of a large variety of lymphocytes from donors of known HLA phenotype or the in vitro cultivation of a large number of HLA-typed cell lines, is therefore complex and laborious to carry out.
More sensitive techniques are also known, such as immunoenzymatic assay on a solid substrate (“ELISA” for “Enzyme-Linked ImmunoSorbent Assay”). In addition, there has recently appeared the technique of immunofluorimetry coupled with detection in flow, which is designed on the principle of flow cytometry. The principle of flow immunofluorimetry consists in fixing purified HLA class I or HLA class II antigens to the surface of polystyrene beads. The anti-HLA antibodies which recognise the HLA class I or HLA class II antigens bind to the antigens bound to the surface of the beads and are revealed by anti-IgG secondary antibodies coupled to a fluorescent group after washing of the polystyrene beads. The secondary antibodies are detected by flow fluorimetry. Their fluorescence intensity is additionally quantified.
For screening tests there is used a plurality of types of beads in admixture, each type of beads carrying on the surface a plurality of HLA antigens, either of class I or of class II. Such an approach allows the presence of anti-HLA antibodies to be detected but without permitting the identification of their specificity/specificities.
In order to identify and characterise the specificity of the antibody, on the other hand, there is used a plurality of types of beads in admixture, each type of beads carrying on the surface a single HLA antigen.
Kits for the detection and identification of anti-HLA antibodies are known. They comprise polystyrene beads coated with HLA class I antigens or HLA class II antigens, and polystyrene beads coated with human IgGs. Also marketed are an anti-human IgG secondary antibody coupled to phycoerythrin, and a serum without anti-HLA antibodies as negative control. Such a negative control is suitable for quantifying the non-specific fixing of the secondary antibody to the polystyrene beads. Such kits do not comprise a positive control, or a sensitivity control, or a standard allowing the concentration of anti-HLA antibodies (expressed, for example, in mole/l or in g/l) in the analysed medium to be derived precisely from the measured fluorescence intensity.
In addition, such kits without a calibration and/or sensitivity control do not allow the sensitivity threshold of the analysis method to be determined, that is to say the minimum value of the signal that makes it possible to affirm that the signal observed is significantly greater than the background noise of the measurement.
In order to remedy this lack of a positive control in methods for the screening and/or quantification of anti-HLA antibodies, immunology and histocompatibility laboratories use, as positive control, a mixture of several serums of several individuals immunised against several HLA antigens.
In such a positive control, the concentration of each of the antibodies of the serums of the immunised individuals is unknown. Such a mixture of serums does not allow the intensity of the fluorescence measurement to be correlated with a concentration (mol/l or g/l) of a specific antibody of the mixture of serums. It therefore does not allow the antibodies present in the serum of the transplant patient or patient awaiting transplant to be quantified. It therefore also does not allow the real risks of the occurrence of a hyperacute humoral response to be evaluated.
The reactivity of such a mixture of serums is variable from one antigen to another, and their use does not allow the detection threshold to be fixed for each HLA antigen studied. Moreover, such a mixture of polyclonal antibodies obtained from patient serums is available in a limited quantity and is quickly exhausted. It must therefore be replaced by a different mixture, which is also available in a variable quantity, which does not allow said mixture to be exchanged between laboratories with a view to standardisation of the results. The variability of the mixtures of serums from one batch to another requires frequent validation of the batches, which are neither comparable nor reproducible from one batch to another.
Using such a mixture of serums, the inventors have shown (FIGS. 2, 3 and 4) that the fluorescence intensity value associated with each type of polystyrene beads depends on the nature of the HLA class I or class II antigen carried by each of the types of polystyrene beads. In addition, the fluorescence intensity value associated with each of the types of polystyrene beads shows considerable variability over time, especially over a period of approximately five months. That value varies (FIG. 4, hatched histograms) between 1000 and 20,000 average fluorescence units.
As a result, the average value of the fluorescence intensity measured on all the polystyrene beads exhibits a considerable dispersion (calculated by its standard deviation), which does not allow the anti-HLA antibodies in the liquid medium to be quantified. Such a dispersion is shown in FIG. 4 (hatched histograms) of the present patent application, which is given to illustrate a standard of the prior art.
Such a dispersion of the fluorescence intensity measurements does not allow a distinction to be made (in particular for low fluorescence intensity values) between a fluorescence intensity value which is low but reflects the presence of a low concentration of anti-HLA antibodies, and a low fluorescence intensity value which cannot be distinguished from the background noise of the measurement.
For the same reasons as set out above, such a preparation used as a positive control in the prior art does not allow the concentration of antibodies present in the liquid medium, especially in a serum collected from a transplant patient or a patient awaiting a transplant, to be determined precisely.
The invention aims to remedy these disadvantages by proposing a monoclonal chimeric immunoglobulin as a standardisation and positive control and sensitivity reagent in the serological analysis of anti-HLA class I antibodies or anti-HLA class II antibodies, especially within the context of organ transplantation.